Human Glyco-engineered CHO Cells Abstract Glycosylation is well known to modulate the activity of therapeutic proteins. Recently, sialylation of the N-linked glycan chains has been shown to significantly affect recombinant glycoprotein stability and activity. For example, the degree of sialylation directly correlates with serum half-life. Sialic acids are also important modulators of immunogenicity, a major problem with protein-based therapeutics. Immunogenicity inversely correlates with the degree of sialylation. Negatively charged sialic acids influence protein-specific parameters like thermal stability, resistance to proteolysis, and solubility. Despite the importance of sialylation, sialic acid incorporation by the production platforms in wide use today, including Chinese hamster ovary (CHO) cells, is inefficient and highly variable, a product consistency issue. Variable sialylation is due largely to the activity of sialidases, as is the incorporation of the non-human sialic acid Neu5Gc, to which most humans possess antibodies. Therefore, it is of great interest to develop methods or platforms to yield uniformly sialylated proteins with a fully human sialylation profile. While several methods have been described to address sialic acid incorporation, none has yet been widely adopted due to inefficiencies or the increased cost of production. We have recently modified the CRISPR/Cas system for genome engineering to develop a method for rapid, efficient generation of homozygous negative cell lines: Double-allele Knockout (DAKO) technology. We will apply DAKO to delete sialidase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) in CHO cells. Antibodies and other glycoproteins produced in these cells will have a high degree of sialic acid incorporation and will be free of Neu5Gc.